Summary. Tyrosine phosphorylation of proteins in cell-ECM adhesions is a major factor driving the proliferative growth, motility, and invasion of cells. Integrin-mediated adhesion activates a signaling complex formed by the association of two tyrosine kinases: FAK and Src. A major substrate of the FAK/Src complex is the docking protein p130CAS, which undergoes phosphorylation on numerous tyrosine residues in its "substrate domain". Recent studies demonstrated the importance of FAK/Src/CAS complex signaling in the related processes of cell motility and invasion. The broad long-term objective of this project is to fully understand the molecular interactions necessary for FAK/Src/CAS complex activation and how downstream signaling events impact the dynamic events that underlie cell motility and invasion. Three specific aims are proposed. Aim 1 will use live cell imaging of fluorescent-tagged CAS variants to determine the dynamics and mechanism of CAS adhesion site targeting. The identification of CAS-interacting proteins important for its localization will also be pursued. Aim 2 will employ time-lapse microscopy to determine the impact of CAS substrate domain phosphorylation on the dynamic processes of lamellipodial protrusion and adhesion assembly/disassembly that drive cell motility, and on the dynamic assembly of podosomes-invadopodia that drive invasion. In related studies, the capacity of CAS substrate domain phosphorylation to promote actin polymerization will be investigated. While the first two Aims make use of CAS-null mouse fibroblast models, Aim 3 extends the work to model invasive human breast cancer cell lines through the use RNA interference to disrupt CAS expression. The impact of the CAS knockdown on cell proliferation and invasion in vitro and on tumor growth and metastasis in vivo will be assessed. Reconstitution experiments with wild-type vs. CAS mutational variants will reveal the important functional domains. Relevance. The proposed studies will fill key gaps in our understanding of the basic signaling machinery that drives the complex dynamic events important for cell motility and invasion. These events have a central role in normal tissue development/regeneration and in many pathological processes including arthritis, atherosclerosis, inflammation, osteoporosis, and cancer progression. The work has potential to reveal new therapeutic targets and prognostic indicators for diseases that arise from aberrant motility and invasion.